5-methylcytosine, present at 70-80% of all CpG dinucleotides, is the only normal modified base found in mammalian DNA. It has been known for more than two decades that the level of 5-methylcytosine bases is significantly reduced in tumor tissues relative to normal tissues (Feinberg and Vogelstein 1983; Gama-Sosa, Slagel et al. 1983; Riggs and Jones 1983). Later it was observed that gene-specific hypermethylation events at CpG-rich, so-called CpG-island sequences occur in cancer tissues (Baylin, Hoppener et al. 1986). In the 1990s researchers reported hypermethylation of CpG islands of several known and putative tumor suppressor genes and other genes involved in important genome defense pathways such as DNA repair (Gonzalez-Zulueta, Bender et al. 1995; Herman, Merlo et al. 1995; Merlo, Herman et al. 1995; Kane, Loda et al. 1997; Costello and Plass 2001; Esteller, Corn et al. 2001; Jones and Baylin 2007). Today, there are many reports that have documented methylation of CpG islands associated with a large number of different genes, including almost every type of human cancer. In lung cancer, several specific CpG islands are methylated including those associated with CDKN2A, RASSF1A, RARbeta, MGMT, GSTP1, CDH13, APC, DAPK, TIMP3, and several others (Dammann, Li et al. 2000; Zochbauer-Muller, Fong et al. 2001; Yanagawa, Tamura et al. 2003; Topaloglu, Hoque et al. 2004; Dammann, Strunnikova et al. 2005). The methylation frequency (i.e. the percentage of tumors analyzed that carry methylated alleles) in the published studies differs widely depending on the histological type of tumor, the study population, and/or the methodology used to assess methylation.
As aberrant methylation (e.g., hypermethylation) of CpG islands is a phenomenon commonly observed during the development and progression of human tumors, detection of methylated CpG islands in easily accessible biological materials or samples such as serum, urine or sputum has the potential to be useful for the early diagnosis of cancer including lung cancer (Laird 2003; Belinsky 2004; Ushijima 2005). Therefore, there is a need to identify CpG islands containing biomarkers that would have specificity in discriminating disease (e.g., tumor) from normal tissue and are aberrantly methylated during the onset or developing or remission stage of the disease.